Phosphonate monoesters and method of preparation

Abstract

Synthesis of phosphonate monoesters, as well as the use of such monoesters as substrates for enzymes which normally hydrolyze phosphodiester linkages, and a method for distinguishing between Type I and Type II phosphodiesterase enzymes using such monoesters as substrates are disclosed herein. Monoesters of phosphonic acids are prepared by the displacement of chloride from an appropriate phosphonic acid dichloride by a phenol or alcohol with pyridine as the solvent, reacting the same and then removing the solvent, after which the remaining ester chloride and dichloride are hydrolyzed by addition of water. The acid and ester are then separated and the acid-free ester is taken up in a polar organic solvent and precipitated as a salt by the addition of aqueous base, with the product then being filtered, washed with fresh acetone, dried and then stored until needed. The thus formed phosphonate monoesters are shown to be effective for use as substrate for phosphodiesterase of the Type I specificity and are shown to be ineffective for use as substrates for phosphodiesterase of the Type II specificity. The type of phosphodiesterase is therefore determined by the amount of activity against phosphonate monoesters.

What is claimed is: 
     
       1. A substrate for phosphodiesterase enzymes, said substrate being a biologically active phosphonate monoester that is hydrolized by said phosphodiesterase enzymes with said phosphonate monoester being selected from the group consisting of 4-nitrophenyl phenylphosphonate, 2-naphthyl phenylphosphonate, 4-nitrophenyl cyclohexylphosphonate, 2-naphthyl cyclohexylphosphonate, 4-nitrophenyl 1-propylphosphonate, 2-naphthyl 1-propylphosphonate, 4-nitrophenyl 2-propylphosphonate, 2-naphthyl 2-propylphosphonate, 4-nitrophenyl chloromethylphosphonate, 2-naphthyl chloromethylphosphonate, and 2-naphthyl methylphosphonate. 
     
     
       2. A method for synthesising phosphonate monoesters, said method comprising: providing a phosphonic acid dichloride capable of being reacted to form a monoester monochloride in an aromatic solvent;   adding dimethylformamide in an amount up to about twice that of said phosphonic acid dichloride to said phosphonic acid dichloride in said aromatic solvent to prevent formation of diester;   adding 2-naphthol or 4-nitrophenol in an amount up to about three times that of said phosphonic acid dichloride after the addition of said dimethylformamide so that an interaction occurs with said phosphonic acid dichloride to displace chloride therefrom and form a monoester monochloride;   hydrolyzing the monochloride and any remaining dichloride; and   separating the monoester from the free phosphonic acid.   
     
     
       3. The method of claim 2 wherein said monoester monochloride is formed by reacting the phosphonic acid dichloride and 2-naphthol or 4-nitrophenol with pyridine for a period between 3 and 4 hours at room temperature, and then removing the pyridine on a rotary evaporator. 
     
     
       4. The method of claim 2 wherein a salt is formed by adding an aqueous base to the monoester and precipitating the same by the addition of a polor non-aqueous solvent. 
     
     
       5. The method of claim 4 wherein said aqueous base is concentrated NH 4  OH. 
     
     
       6. The method of claim 2 wherein said phosphonate monoester formed is filtered, washed with acetone, and dried by a vacuum dessicator prior to storage at a temperature below 0° C.